maternal cigarette smoking and the incidence of pre-eclampsia the potential roles of carbon monoxide within this puzzling association

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Maternal Cigarette Smoking and the Incidence of Pre-Eclampsia: The Potential Roles of Carbon

Monoxide within this Puzzling Association By

SHANNON AMBER BAINBRIDGE

A thesis submitted to the Department of Anatomy & Cell Biology in conformity with the requirements for

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To my father:

Your never-ending pursuit of knowledge has inspired me from the time | was a little girl

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ABSTRACT

Pre-eclampsia (PE) is a disorder of pregnancy affecting 5-7% of all pregnancies, characterized by maternal hypertension and proteinuria Interestingly, women who smoke cigarettes throughout gestation are 33% less likely to develop this disorder than non-smoking women The specific mechanisms through which cigarettes confer protection from PE are unknown; however the reduced risk for PE has been strictly associated with the combustible products of tobacco It is postulated that carbon monoxide (CO), a major component of cigarette smoke, may play a vital role in this association as CO has been shown to possess several regulatory and cytoprotective properties The enzyme responsible for CO production, heme oxygenase, has been isolated throughout the human placenta Placental CO production has been linked to trophoblast migration through the uterine wall, spiral arterial remodelling and optimization of placental perfusion As these important aspects of placental development are compromised in PE women it is hypothesized that cigarette smoking reduces the risk of developing PE via CO-mediated cytoprotective effects Specifically, it is proposed that CO is capable of: increasing placental perfusion, thus decreasing localized oxidative stress; decreasing ischemia/re- perfusion induced apoptosis within syncytiotrophoblasts, thus reducing placental debris shedding into the maternal circulation; and maintaining normal maternal endothelial functioning An examination of the above mentioned hypothesis was undertaken through several research objectives Initially a detailed profile of molar CO concentrations observed within the maternal and feto-placental

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circulations of non-smoking, smoking and PE women was completed Using these in-vivo CO concentrations the placental vascular effects of CO were examined using an in-vitro placental perfusion set-up The anti-apoptotic properties of CO were also examined within the syncytium of term placenta following oxidative stress induced injury Finally, maternal endothelial health and functioning was examined, through measurements of soluble guanylyl cyclase activity, using maternal omental vessels collected from non-smoking, smoking and PE women Carbon monoxide was shown to possess potent vasodilatory and cytoprotective effects within the human placenta, at circulating concentrations of CO measured in smoking women The results of this thesis identify potential mechanisms through which smoking women are at a reduced risk of developing PE Furthermore, the therapeutic potential of exogenous CO in the prevention and/or treatment of PE is highlighted

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ACKNOWLEDGMENTS

There are several people who have provided me with guidance, support and love over the past five years and they deserve both recognition and thanks These people include my dear friends and loving family and all those who have provided mentorship throughout my training It is because of these people that | have become who | am today

| would like to first express my heartfelt thanks and appreciation to my family You have been my biggest fans and have encouraged me to follow my dreams, wherever they may take me Mom and Dad, you have given me the gifts of compassion, integrity, curiosity and strong work ethic These tools have played a pivotal role in getting me to where | am and will no doubt be of tremendous importance as | head off to pursue a career in research and academia Roger, you have pushed me to think outside of the box and helped me to recognize that there is much more to this world outside of science While | take great pride in the accomplishments | have completed to date | know that they would not have been possible without the support of my family Thank you

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you | must also thank Dustin; your love, respect and support over the past couple years has been unending You have allowed me to grow emotionally and intellectually Thank you so much for keeping me grounded and putting a smile on my face every day

Further, | would like to thank the people who have provided me with an enriching and stimulating learning environment | would like to’ acknowledge Anne Farley, Richard Casselman and John Dacosta for their tremendous help and technical expertise | would also like to recognize all the members of the Smith lab who made it a pleasure to go into work every day: Liz, Laura, Erica, Michelle and Mark | would like to specifically thank Lindsay Patrick for her support and friendship over the years You rock lady! Thanks must also go to Dr Charles Graham, who continually encouraged me to think creatively and ask questions

Finally, my utmost gratitude goes out to my mentor Dr Graeme Smith Graeme, | cannot imagine having a better supervisor You have provided me with respect, trust, encouragement, and tremendous guidance Your passion for research is truly inspiring and contagious Coming into your lab as a fourth year student | never dreamt that | would be leaving six years later with a doctorate But your remarkable guidance made the choice to pursue a career in research unbelievably easy You have taught me so much, but most importantly you have shown me what it means to be a true mentor This lesson will stay with me for the

rest of my life Thank you from the bottom of my heart

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TABLE OF CONTENTS F1 0 ố ốẽốốốố ốốốốốốốốố iii ACKNOWLEDGMENTS HH ng nen V LIST OF TABLES - LG HH TH ng Họ ng X LIST OF FIGURES HH go 0000080 xi Não cố ố xi 8e 0 i2//0ï9 1n xiii

CHAPTER 1: GENERAL INTRODUCTION .:::cccccsesessssesesersees 1

1.1 Heme Oxygenase System in Pregnancy .c:csccsssseessseessssesssesessscers 2 1.1.1 Heme oxygenase nh TT ng TT TH ng vàn 2 1.1.2 The HO system as it relates to pregnancy cccccàccerceeee 4 1.1.3 The specific roles of Fe** during pregnancy ¿-¿cccccteterrrkc 7 1.1.4 The specific roles of biliverdin/bilirubin during pregnancy 7 1.1.5 The specific roles of CO during pregnancy .:::cccccssssseeesssseeeeeeeees 9 1.2 The HO/CO System and Complications of Pregnancy - 15 1.2.1 Pre-OClaMPSi€ eee 16 1.2.2 The HO/CO System and PE ceccccccecssseeececessseeeecesenssseesecsssttenees 16 1.3 Puzzling Relationship between Smoking and PE s- 22 1.3.1 Tobacco and Cigarette Smoke TQ TH n HH nhu 22 1.3.2 Smoking and Pregnancy - - c1 1n n nh nn nghe y 23 1.3.3 Smoking and PE cc Q00 0n ng khen Hy 23 1.4 HYPOTHESES AND OBJECTIVES 50c nerverrreerreerrxree 27 1.4.1 Specific Hypotheses ng ng hệt 27

1.4.1.1 Carbon monoxide, at concentrations found in smokers, is capable of increasing placental perfusion and thus decreasing incidences of localized OXidatiV© SỈF@SS HH TT KT KT 27

1.4.1.2 Carbon monoxide, at concentrations found in smokers, is capable of decreasing I/R induced apoptosis in syncytiotrophoblasts and would

hence reduce placental debris shedding into the maternal circulation 30 1.4.1.3 Carbon monoxide, at concentrations found in smokers, is capable of maintaining normal endothelial functioning in the maternal vasculature in the face of decreased NO availability cece cccccccccccceeeeeeeseeeeeeees 31 1.4.2 Specific ObjectiVes LH nh nh tk ke 33

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CHAPTER 2: METHODOLOGIES cookie 35-

2.1 MEASUREMENT OF CO CONCENTRATIONS USING GAS-SOLID

CHROMATOGRAPHY SG HH ng cm 36 2.2 PLACENTAL PERFUSION PREPARATION che 37

CHAPTER 3: MATERNAL AND FETAL CARBON MONOXIDE CONCENTRATIONS cm re 42 ABSTRACT HH Ho 43 INTRODUCTION nọ the, 44 MATERIAL AND METHODS .- Q HH ng như, 47 RESULTS - GHI họ 0 n4 49 DISCUSSION - TH HH HH Tnhh 51 TABLES AND FIGURES - cọ HH to on 57

CHAPTER 4: CARBON MONOXIDE DECREASES PERFUSION

PRESSURE IN ISOLATED HUMAN PLACENTA 62 '\ cài, là .,HHẬHH 63 Dã e0 1 64 MATERIALS AND METHODG c:ssssssssssssessessssssssssssssssessssssesseesseassssesanenseeess 66 n0 1 70 DISCUSSION ccieciereee .,ÔỎ 73 ACKNOWLEDGMENTS .ssssssssssssssssssessesssessessseessesssssnssssessscessssseessessseessessees 77 CHAPTER 5: THE EFFECT OF NICOTINE ON JN VITRO

PLACENTAL PERFUSION PRESSURE - 82

ABSTRACTT -GG cọ nọ hi hi hi tì ni và nh 83

INTRODUCTION - nọ in v 84

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CHAPTER 6: CARBON MONOXIDE INHIBITS HYPOXIA/RE- OXYGENATION INDUCED APOPTOSIS IN SYNCYTIOTROPHOBLASG T :::cccccsssssssseeesesessecsccensssssseesseosenees 97 F1 an 98 INTRODUCTION HH ng gọn nh 99 MATERIALS AND METHODS -QQQQQnnHHn HH ng ng 55 103 RESULTS 110 DISCUSSION HH HH nọ 0n 00050755 08 114 ACKNOWLEDGMENTS LH HHnHg HH ng nhan 119 FIGURES 120

CHAPTER 7: GENERAL DISCUSSION - << 128

Identification of CO as a mediator in the reduced risk of PE amongst Cu SG ư 129 Identification of CO as a therapeutic agent in the prevention and 06-2111-1820 ¬a ố na 137 ORIGINAL CONTRIBUTIONS - QQ GGQ H n khh 142 FUTURE STUDIES ng nVRp 144

APPENDIX A: RESEARCH PROPOSAL AND SAMPLE DATA

FROM MATERNAL ENDOTHELIAL FUNCTION STUDY 149

REFERENCE LIST .- - - HH vn 156

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LIST OF TABLES

TABLE 1.1 The potential maternal, placental and fetal effects of cigarette

smoking during pregnancy - - che 26

TABLE 3.1 Molar concentrations of CO measured in maternal and umbilical circulations along with end-tidal breath CO concentrations from non-smoking normotensive, smoking normotensive, non-

smoking pre-eclamptic and smoking pre-eclamptic women at term 59

TABLE 5.1 Fetal arterial perfusion pressure (+ STDV) recorded using the in vitro dually perfused cotyledon preparation following perfusion with

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LIST OF FIGURES

FIGURE 1.1 Heme degradation pathway and physiological roles of its breakdown products CO, Fe2+ and biliverdin during a healthy and PE

s/-le)a <2 .ố.ố 14 FIGURE 1.2 The current working model for the progression of pre-

eclampsia, demonstrating possible sites of action and mechanisms through which CO from cigarette smoke may be capable of halting the

progression of the diSOrde@r HH HT ke xe 34 FIGURE 2.1 Schematic diagram of CO measurement using gas-solid

Chromatography 110150 ằ aa ốằ 39

FIGURE 2.2 Schematic diagram of the open system, dually perfused

9i-te-)á)-10eá01-1948 00HO 40 FIGURE 2.3 Cannulation of fetal artery, fetal vein and placement of

maternal artery and maternal vein in the dually perfused cotyledon

I9-9-1/-\1sà NAaaaiaadididid 41 FIGURE 3.1 Comparison of CO concentrations in maternal and umbilical

cord blood in NON-SMOKEFS VS SMOKELS ccccccccceceeeeeesseseseeeeeeseeeeeeaaaaes 58

FIGURE 3.2 Carbon monoxide concentrations in umbilical arterial vs

umbilical venous bÌOod - HT TH TH ng nu ng nhu 60

FIGURE 3.3 Circulating CO concentrations in normotensive (+/- smoking)

VS PE (+/- smoking) WOmen - c1 c1 TH H HH net 61 FIGURE 4.1 Vascular effects of CO in isolated perfused cotyledon 79 FIGURE 4.2 Vascular effects of CO in isolated perfused cotyledon in the

presence of the SGC inhibitor ODQ 00.0.0 ceeescccccesssceeteeseeessseteeeserssesreeerens 80 FIGURE 4.3 Vascular effects of CO in isolated perfused cotyledon in the

presence of the SGC potentiator YC-1 0.0 ccccessesssssesssseeeeeeseeeesssaeaes 81 FIGURE 5.1 Vascular effects of nicotine in isolated perfused cotyledon 96 FIGURE 6.1 Schematic of H/R model employed to assess anti-apoptotic

action of CO within the syncytiotrophoblasts cccscesesesssseeeeeeeeeesees 121

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FIGURE 6.2 Immunoflourescent images demonstrating CO mediated

inhibition of H/HR induced apoptosis in syncytiotrophoblasts 122 FIGURE 6.3 Quantification of CO mediated inhibition of H/R induced

apoptosis in syncytiotrophoblas†s - L TH nen HH key 123 FIGURE 6.4 Quantification of p85 fragment of PARP in untreated and CO

treated explants following an H/R insult cà 124 FIGURE 6.5 Morphological analysis of untreated and CO treated villous

explants following an H/R insult and comparison to morphology

observed in villous explants from PE placentae eee cece eee eeees 125 FIGURE 6.6 Quantification of sGC activity in untreated and CO treated

villous explants following an H/R insult nhe 127

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LIST OF ABBREVIATIONS ANOVA: analysis of variance Ca””: calcium CAT: catalase cGMP: cyclic guanosine monophosphate CO: carbon monoxide CO;: carbon dioxide

DAPI: 4',6-diamidino-2-

phenylindole DMSO: dimethy! sulfoxide ELISA: enzyme linked

immunoassay EM: electron microscopy FA: fetal artery

Fe**: Free Iron FV: Fetal Vein g: gram GPx: glutathione peroxidase GTN: Glyceryl Trinitrite H/R: hypoxia/re-oxygenation H2Oz2: hydrogen peroxide xii Hg: mercury

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mM: millimolar mmHg: millimetre of mercury mRNA: messenger ribonucleic acid MV: maternal vein No: nitrogen NAD: nicotinamide adenine dinucleotide

NADH: nicotinamide adenine

dinucleotide with hydrogen NaHCOs: sodium bicarbonate ng: nanogram

nm: nanometer nM: nanomolar NO: nitric oxide

NOS: nitric oxide synthase Oz: oxygen O*: superoxide ODQ: 1H-(1,2,4)oxadiazole(4,3- a)quinoxalin-1-one p: probability PARP : poly-ADP ribose polymerase PBS: phosphaie-buffered saline PE: pre-eclampsia XIV PFA : paraformaldehyde pmol: picomole

pOz: partial pressure of oxygen ppm: parts per million

ROS: reactive oxygen species RT-PCR: reverse transcription-

polymerase chain reaction SEM: standard error of the mean SGA: smail for gestational age sGC: soluble guanylyl cyclase SOD: superoxide dismutase STBM : syncytiotrophoblast micro- fragments STDV: standard deviation TCA: trichloroacetic acid TdT : terminal deoxynucleotidyl transferase TGF-B : transforming growth factor beta

TNF- a: tumor necrosis factor alpha TUNEL: terminal deoxynucleotidy|

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"UA: umbilical artery UV: umbilical vein vs.: versus

YC-1: 3-(5-hydroxymethyl-2 -furyl)-1 - benzylindazole

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Chapter 1

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1.1 HEME OXYGENASE SYSTEM IN PREGNANCY

1.1.1 Heme oxygenase

The enzyme heme oxygenase (HO) was originally characterized in 1968 by Tenhunen et al who described it as the mediator of heme metabolism in a cell ' This led to the notion that the enzyme was a housekeeping protein involved in maintaining homeostasis of the heme pool A number of studies have since been conducted in the area of heme metabolism and HO in particular It is now

recognized that HO is involved in the control of vascular tone **, regulating anti- inflammatory *’ and anti-apoptotic °° responses as well as reducing oxidative stress and subsequent tissue damage in several organ systems '*'* These

various properties attributed to HO are carried out through the catalytic products of heme degradation, namely carbon monoxide (CO), biliverdin and free iron

(Fe**) (Figure 1.1.)

Three isoforms of the HO protein have been identified HO-1 is a 32 KD inducible form of the enzyme; HO-2 is a 36 KD constitutive form of the enzyme

while HO-3 is the least characterized and least active of the isoforms '* '* HO-1

is ubiquitously distributed in mammalian tissues, although expressed in high concentrations in areas of high erythrocyte turnover such as the spleen and liver

"15 HO-1 protein expression and activity is induced by several stimuli including

heme, metalloporphyrins, transition metals, cytokines, endotoxins, hyperthermia,

13, 15

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is found in numerous tissues throughout the body, and appears to be involved in

maintenance of basal heme metabolism '* '* The vast array of activating stimuli

coupled with HO's hemodynamic, anti-oxidant, anti-inflammatory and _ anti- apoptotic properties has led to questions regarding the functional significance and role of this enzyme under both physiological and pathological circumstances

All three catalytic products of heme degradation, CO, biliverdin and Fe", were originally deemed toxic compounds; at high concentrations all three of

these compounds poses cytotoxic properties '*'® However, it is now recognized

that at concentrations produced through the actions of HO, all three compounds in fact have significant cytoprotective properties

Carbon monoxide has been demonstrated to display several of the same physiological functions as its diatomic cousin nitric oxide (NO) These include

decreasing vascular tone via vasodilation * '°, inhibition of platelet aggregation 7°

as well as inhibition of the apoptotic and inflammatory cascades ° ” ° Promising

studies are now emerging concerning the therapeutic potential of CO in reducing organ transplant rejection

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and third catalytic products of heme degradation (biliverdin and Fe**) also contribute to HO’s anti-oxidant effect Biliverdin and its subsequent breakdown

21 A recent

product bilirubin demonstrate potent antioxidant characteristics

description of the cycle in which biliverdin is reduced to bilirubin, via biliverdin reductase, and then is subsequently recycled back into biliverdin suggests a mechanism through which the anti-oxidant properties of these two molecules

may be amplified in vivo ** Free iron, released from the core of the heme

molecule, is capable of extensive cellular damage; through the Fenton reaction, it

is capable of promoting the generation of damaging free radicals ** *“ The

25

enzyme HO, however, interacts with intracellular iron pumps as well as

upregulating the generation of ferritin 7° , a potent iron-chelating molecule Therefore, while heme catabolism will initially increase levels of free iron, the additional actions of HO have the net effect of decreasing intracellular free iron and thus limit the generation of ROS The antioxidant properties of both biliverdin/bilirubin and ferritin have been used therapeutically to ameliorate

hepatic injury in several models, including ischemia/reperfusion injury 2” 78 1.1.2 The HO system as it relates to pregnancy

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in placental function °° In order for this enzyme to be recognized as a significant contributor to normal placental function three criteria must be fulfilled:

e HO must be localized in placental tissue e HO must be functional within the placenta

e HO by-products must be capable of exerting physiological effects on the various placental and/or fetal tissues

Identification and localization of HO protein within the placenta has been studied extensively The mRNA for both HO-1 and HO-2 isoforms have been measured in placental tissue homogenate using RT-PCR, demonstrating

elevated concentrations of HO-2 mRNA compared to that of HO-1 % %7 | n addition, both placental HO-1 and HO-2 mRNA increase with advancing gestation °°" The HO proteins have been identified using western blot analysis in chorionic villi, chorionic plate, basal plate and fetal membranes HO-2 protein

content was again found to be higher than HO-1 in all areas examined °° %: 9%,

The differential expression of the various HO isoforms throughout the placenta is also characterized using immunohistochemical staining A wide distribution of placental HO is observed in syncytiotrophoblasts, endothelium and smooth muscle cells of umbilico-placental blood vessels as well as fetal membranes Early in pregnancy, HO-2 appears in abundance in the syncytiotrophoblasts,

31, 33

cytotrophoblast cell columns and vascular endothelium , while at term there is wide distribution amongst the endothelial and vascular smooth muscles cells of

31

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intravascular and extravillous interstitial trophoblast cells *° In all studies, a decrease in HO-1 staining was observed in every area examined compared to HO-2 staining However, the HO-1 enzyme was identified in syncytiotrophoblast

and cytotrophoblast cells as well as the vascular endothelium °° 3": °%,

With the presence of HO protein in the placenta, activity of HO under physiological conditions was examined Based on the rate of CO formation from exogenous heme, HO activity was found to be highest in the chorionic plate,

chorionic villi and basal plate *4 Enzymatic activity was also measurable in the

umbilical artery and vein *° Recently the effects of both oxygen and glucose availability on HO activity in the placenta were examined and it was determined that although an increase in HO-1 mRNA and protein expression was seen in response to hypoxia, the overall HO enzymatic activity decreased under hypoxic conditions °° The opposite was true in the case of glucose, where a 24-hour pre- incubation in a glucose-deficient medium resulted in both an upregulation of HO expression and activity °” These two studies indicate that the regulation of the HO system in the placenta is complex and in part reliant upon local glucose and

oxygen concentrations

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1.1.3 The specific roles of Fe** during pregnancy

lron is an essential nutrient, associated primarily with heme containing proteins throughout the body Pregnancy is a time of heightened iron demand in order to accommodate for increases in blood volume, placental development and fetal growth *° Iron deficiency throughout pregnancy is quite common, affecting 18% of pregnancies in developed countries and up to 88% for women of low

socioeconomic status * lron deficiency has been linked to reductions in

placental capillary surface area °°, with potential impacts on nutrient transfer to

the fetus; severely anemic women are at an increased risk of delivering intra-

uterine growth restricted infants *° *’ Therefore, it is not surprising to find an

enzyme capable of mobilizing heme-bound iron throughout the placenta While the primary protective functions of HO may be carried out through the actions of CO and biliverdin/bilirubin, the ability of HO to make available a nutrient essential to placental and fetal development no doubt adds to its importance during pregnancy Little work, however, has been completed to date examining the

we

specific roles of endogenous iron storage and release on placental function

1.1.4 The specific roles of biliverdin/bilirubin during pregnancy

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oxidative stress as most reactive oxygen species are membrane permeable and capable of crossing the placenta ** In addition, while total lipids in the fetal circulation are lower than that of an adult, a higher proportion of polyunsaturated fatty acids have been measured in umbilical cord plasma compared to adult plasma “° This type of fatty acid is particularly susceptible to oxidation, placing the fetus at potentially higher risk of oxidative injury Despite the increased risk for oxidative damage in the feto-placental unit, in healthy pregnancies this damage does not occur This would indicate that antioxidant system(s) must be in place to protect both placental and fetal tissues from this sort of insult Various enzymatic anti-oxidant systems, such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), have been characterized within the

placenta 4447, Currently the roles of non-enzymatic scavengers, such as œ- Tocopherol (Vitamin E) and ascorbic acid (Vitamin C), bilirubin and ferritin, are being studied as potentially important anti-oxidants within the placenta These non-enzymatic antioxidants prevent lipid peroxidation by trapping oxygen free radicals and breaking the peroxidation chain reactions *° Within human placental and fetal tissues, concentrations of the non-enzymatic antioxidants

bilirubin/biliverdin are shown to increase with advancing gestation “7 This is

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Gopinathan et al indicates that in neonates a very strong correlation between bilirubin levels and total plasma antioxidant activity exists °°, further supporting biliverdin/bilirubin playing a pivotal role in maintaining a homeostatic redox balance during pregnancy

1.1.5 The specific roles of CO during pregnancy

Recent studies have focused on the vascular effects of CO in the placenta It is recognized that term arterial vessels of the feto-placental circulation are maintained in a state of near maximal dilation in order to facilitate oxygen and

nutrient delivery °' The placenta lacks innervation ** and therefore is dependent

upon locally produced and circulating vasoactive compounds for hemodynamic control With the abundance of HO protein found throughout the human placenta, coupled with the known vasodilatory properties of CO in other organ systems, it was hypothesized that CO was involved in maintaining placental vascular tone

33

throughout gestation Using the dually perfused cotyledon preparation, inhibition of HO activity by zinc protoporphyrin resulted in increased placental

perfusion pressure °° indicating a role for CO in the maintenance of basal vascular tone within the placenta Further, it has been demonstrated that CO is

capable of relaxing pre-constricted anchoring villi °° This would indicate that HO

derived CO may not only increase intra-placental fetal perfusion by dilating placental blood vessels but it may also enhance intervillous volume and blood

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The role of CO in the maintenance of uterine quiescence throughout pregnancy and the onset of labour warrants further investigation This potential role of CO parallels the ongoing debate regarding the role of NO in the maintenance of uterine quiescence and its potential use as a tocolytic agent ** Myometrial quiescence during pregnancy has been characterized by an increase

in the concentrations of myometrial cGMP °° Nitric oxide, as well as CO,

stimulates soluble guanylyl cyclase (SGC) which increases intracellular levels of

cGMP, subsequently relaxing smooth muscle (Figure 1.1)°° Several animal

studies have suggested a role for this NO-cGMP pathway in myometrial

57,58 However debate

quiescence during pregnancy and in the onset of labour

over the presence of nitric oxide synthase (NOS) within human myometrial tissues has led to controversy over whether NO is locally produced and hence capable of acting on myometrial smooth muscle cells in vivo °° Due to similar actions on sGC as NO, CO has now come into the spotlight as a potential mediator of this function during pregnancy Acevedo et al report a 16-17 fold increase in the expression of the HO-1 and HO-2 proteins in pregnant myometrial

tissue compared to non-pregnant myometrium °° Expression of these two proteins has also been reported to increase throughout gestation in both the intravascular and extravillous interstitial trophoblasts within placental bed biopsies °° These results are in contrast to findings by Barber et al who reported no measurable HO-1 protein in non-pregnant or pregnant myometrial tissue, with similar expression of HO-2 between the two groups °° In both studies similar

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HO-1 and HO-2 antibodies were used, the study groups were similar in gestational age and all myometrial biopsies were reportedly taken from similar portions of the uterus It is therefore surprising to observe such contrasting results It is important to note that neither study indicated whether external sources of HO upregulation, such as maternal smoking status °', were similar between the study and control groups Both studies had relatively low sample numbers (n=4/group in the Acevedo study, n=10/group in the Barber study) therefore external confounding factors may play a significant role in the studies outcomes It is also possible that varying results were due to technical differences in methodologies employed by these groups It is clear that replication of these studies, with clearly delineated study populations and widely accepted methodologies, must be undertaken to better understand the expression of the HO enzyme system in pregnant myometrial tissue Regardless of myometrial HO protein expression during pregnancy, the potential role of CO as a tocolytic agent still remains Acevedo and Ahmed reported hemin, a potent HO inducer, was capable of inhibiting both spontaneous and oxytocin-induced myometrial contraction ®° Further support of CO’s potential uterine quiescent capabilities has been obtained through measurements of maternal end tidal CO concentrations These results indicated that women experiencing either pre-term or term uterine contractions had significantly lower levels of end tidal CO, suggesting a possible association between decreased intrinsic CO production and increased uterine activity © Further studies examining the localization of the

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HO-CO system within the pregnant uterus along with further investigation into CO’s uterine quiescent properties are required

Trophoblast migration through uterine tissue and subsequent spiral arteriole remodelling is essential for the development of a healthy placenta and fetus This ability of trophoblast cells may relate to the ability of local autocoids (i.e

63 66

progesterone °°, estradiol ©, angiotensin II ©) to stimulate NO production

Failure to generate adequate amounts of trophoblast NO may predispose women to pre-eclampsia, a disorder of inadequate placentation °’ Therefore it is proposed that NO is required for proper trophoblast differentiation and invasion The localization of HO isoenzymes in extravillous trophoblasts coupled with data indicating that cultured first trimester trophoblast cells are capable of CO production, and the underlying similarities between NO and CO, have led to the hypothesis that trophoblast derived CO may facilitate trophoblast penetration into the spiral arteries early in pregnancy °

A healthy pregnancy is characterized by an underlying systemic inflammatory response that must be continually monitored and regulated by various enzyme systems in both maternal and feto-placental tissues °° Recently, CO has been found to have anti-inflammatory properties in various organ systems including the heart and liver Little work has been completed examining the role of the HO-CO system in maintaining a healthy balance of inflammatory/anti-inflammatory responses throughout gestation The role(s) CO may play in pathological

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disorders associated with an increased inflammatory response have been considered and the HO-CO system will be discussed in relation to pre-eclampsia

below

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NADPH NADP 0, HO Biliverdin Reductase ? Ferritin Bilirubin Relaxation of Inhibit

vascular inflammation Antioxidants smooth muscle and apoptosis

Maintenance of Inhibition of ƒ Maintenance ofa adequate blood excessive : healthy oxidant-

flow through activation of ƒ ' antioxidant uterine and inflammatory | balance within

placental blood and apoptotic | _ the placenta

vessels cascades in -

placental tissue

Decreased and Heightened : {Increased levels oscillating blood activation of = £ of ROS coupled flow through inflammatory | with a decrease in placental blood and apoptotic =F antioxidant

vessels cascades in / capacity within

placental tissue l the placenta

Figure 1.1 Heme degradation pathway and physiological roles of its breakdown products CO, Fe2+ and biliverdin during a healthy and PE pregnancy

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1.2 THE HO/CO SYSTEM AND COMPLICATIONS OF PREGNANCY

Heme oxygenase and its catalytic products may play a significant role in the progression of a healthy pregnancy to term Since this enzyme is linked to several vital tasks of pregnancy such as placentation, placental hemodynamic control and antioxidant protection, the activity or dysfunction of this enzyme is the target of investigation in several disorders of pregnancy While there is a paucity of literature on this topic, there is evidence linking a decreased expression of the HO-1 and HO-2 enzyme at the feto-maternal interface in a model of spontaneous abortion in mice °° Furthermore, studies of first trimester human placental tissue have indicated a significant decrease in HO-2 expression in the invasive trophoblast, endothelial and syncytiotrophoblast cell populations from patients with spontaneous abortion ”° A decreased expression of HO-2 has also been linked to fetal growth restriction; using immunohistochemistry, a decrease in HO- 2 staining was observed in the endothelial cells from term placenta of infants diagnosed with fetal growth restriction ’' Further strengthening the importance of HO in reproductive health, HO-1 null mice are reportedly sterile The bulk of the work completed to date has focused on the pivotal roles that this enzyme system may play in the syndrome of pregnancy known as pre-eclampsia (PE)

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1.2.1 Pre-eclampsia

Pre-eclampsia (PE) is a hypertensive disorder of pregnancy, affecting between 5-7% of all pregnancies It is a leading cause of maternal and fetal/neonatal morbidity and mortality world wide ” It is hypothesized that the progression of PE begins with shallow trophoblast invasion and subsequent

inadequate spiral arteriole remodelling “7 Consequently, the placenta is not effectively perfused and localized areas of oxidative stress are established ”°,

Hung et al have demonstrated that syncytiotrophoblasts lining the intervillous space are at an increased risk of apoptosis and necrosis under the influence of oxidative stress, leading to the shedding of syncytiotrophoblast cellular debris, known as_ syncytiotrophoblast microfragments (STBMs), into the maternal circulation ’° Women with PE have been shown to have an increased load of

STBMs in their circulation ”’ This increased load of placental debris elicits a

heightened maternal inflammatory response and _ vascular endothelial dysfunction The compromised endothelial functioning is directly responsible for the characteristic signs and symptoms used to diagnose this disorder, namely

elevated maternal blood pressure and proteinuria ’ ’* ’° To date, there is no

available cure for this disorder apart from timely delivery of the fetus and more importantly the placenta, which may result in significant iatrogenic prematurity and all its associated complications

1.2.2 The HO/CO System and PE

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Several groups have determined HO expression, localization and activity in the PE placenta with conflicting results to date One study examining levels of HO mRNA using RT-PCR found no significant difference in levels of transcript for either isoform of the enzyme between PE and normotensive placentae ° However, this same study reported a reduced expression of HO-1 protein in PE placentae using western blot analysis, which suggests a translational blockage of this isoform ° This contrasts the results of two other studies examining HO-1 protein content in the PE placenta, one of which reported an increase in protein expression in chorionic villi and fetal membranes *, while the other indicated that the HO-1 protein was undetectable in the placental homogenates collected from both the PE and normotensive placentae ’' The results between the two groups were similar when examining protein expression of HO-2, with no significant changes in HO-2 protein expression being reported in the placenta of PE women

32,71 Barber did, however, report a reduction in HO-2 immunohistochemical

staining in the endothelial cells of the PE placentae ”’ McLaughlin et al characterized HO activity in different placental regions (chorionic plate, basal plate, chorionic villi and fetal membranes) and found no difference in the

32 These

enzymatic activities between PE and normotensive placentae

contradicting results add confusion to HO’s potential role in pathogenesis of PE However variability amongst individual patients, disease state and experimental protocols may explain some of these differences All groups noted large variability in HO protein expression, localization and activity between patients

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Larger sample sizes are required in order to adequately address this question There were also differences in the HO antibodies used in the various

5, 71 while others were

experiments, some being targeted to rat HO protein

targeted to human HO protein * which could result in varying specificity of these

antibodies to human placental tissues and hence varying results In addition, while ail of the studies investigating HO protein expression in the placental tissue used the same criteria for the diagnosis of PE, the studies did not report gestational age or disease severity at the time of tissue collection Furthermore, HO enzyme expression and activity may vary across the placenta Consequently, it is critical that the sampling site be identified and considered when comparing contradictory data

As previously mentioned, while HO expression has been shown to be upregulated under hypoxic conditions, HO enzymatic activity appears to

decrease with decreasing pO2 *° Pre-eclamptic placentae are known to have

increased areas of hypoxia, leading to areas of necrosis or infarct Infarct regions are present in placentae from uncomplicated pregnancies as well, however infarcts in placentae from PE pregnancies tend to be larger and more numerous 78 Lash et al examined the effect of placental tissue damage on HO expression and activity in both PE and normotensive placentae They reported no significant differences in HO-1 protein levels in healthy and infarcted tissue for both normotensive and PE placentae; however they did report that HO-2 protein levels were decreased in the peri-infarct and infarcted villi of PE placentae Using

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immunohistochemical analysis they detected decreases in both HO-1 and HO-2 protein expression in all damaged tissue They further compared HO enzymatic activity in microsomes isolated from morphologically normal and _ infarcted chorionic villi and found decreased HO activity in the damaged tissue under

optimized conditions ®° The results of this study indicate that the ability of

chorionic villi to oxidize heme into CO, biliverdin and Fe** may be compromised in areas of tissue damage in the placentae of women with PE Considering that the proportion of damaged or infarcted tissue is higher in a PE placenta compared to a healthy placenta, it is postulated that the PE placental unit would have lower overall HO enzymatic activity and hence produce lower levels of its three catalytic products compared to a placenta from a healthy woman If one were to imagine a disorder that would present following a reduction in HO enzymatic activity during gestation, based on the arguments provided above for the regulatory and cytoprotective roles of HO’s catalytic products, a resemblance to the disorder now recognized as PE becomes apparent (Figure 1.1.)

It has been proposed that an initial immune response or rejection of fetal tissue may be responsible for the shallow invasiveness and impaired remodelling of spiral arterioles by trophoblast cells in PE ™ °' Several studies have demonstrated an infiltration of activated macrophages or foam cells into the

maternal spiral arterioles of the PE placenta ®* *° These activated macrophages

release a number of compounds (i.e tumor necrosis factor alpha, transforming growth factor beta, indoleamine 2,3-dioxygenase) that are capable of decreasing

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trophoblast cell invasion and possibly initiating the apoptotic cascade within

these cells °* ® This inflammatory environment of the PE placenta has been compared to that established with allograft rejections ”* The relevance of this

similarity comes from publications in the areas of transplantation and allograft rejection research These studies demonstrated increased success in organ transplantation when the organs were pre-incubated with CO or had been

genetically modified to display an upregulation in HO ” ® **°°, They indicate that

CO is capable of reducing the inflammatory response and apoptosis characteristic of an allograft rejection °” These transplanted organs appear to be perfused to a much higher degree than the non-treated organs, and the survival

of the recipients was greatly increased ® ® °° This may also be the case in the

placenta It is suggested that inadequate HO enzymatic activity, and subsequently decreased CO availability, would result in the invading trophoblast cells not being provided with sufficient cytoprotection from the anti-invasive and pro-apoptotic signals initiated by the activated macrophages Consequently the trophoblast cells would display shallow invasion of the decidua and inadequate

remodelling of the spiral arterioles ~

The decreased CO availability, resulting from the decreased HO activity, would also hinder normal placental hemodynamic control As vascular control in the placenta is dependant in large part on locally produced vasoactive compounds, the loss of a key vasodilator in this circulatory system could have significant effects on intra-placental perfusion This would exacerbate localized

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areas of hypoxia established via inadequate blood flow through the constricted spiral arterioles This hypoxic insult, coupled with the reduced expression of the antioxidant bilirubin, could potentially tilt the balance of the oxidant-antioxidant capabilities of the placenta towards increased localized oxidative stress As previously discussed this oxidative insult would then be capable of initiating apoptotic cascades within the syncytial lining of the intervillous space ”°, setting in motion the transport of a placental dysfunction into the maternal compartment,

as is observed in the placentae of women with PE ™ ”°

There have been no studies examining the levels of HO expression and activity in the circulatory system of women who develop PE The results of such studies may provide further insight into the progression of PE from a disorder of placental dysfunction into a disorder of maternal endothelial dysfunction Pre- eclamptics have been shown to have increased levels of platelet and neutrophil activation which promotes vascular damage and obstruction, leading to tissue

ischemia and further tissue damage °° In the human case of HO-1 deficiency,

the most pronounced pathological phenotype is also severe and persistent endothelial damage * While there are certainly several key players in the progression of this disorder, we would argue that a reduction in the HO/CO- Bilirubin system may be of significant importance in the development and progression of PE

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1.3 PUZZLING RELATIONSHIP BETWEEN SMOKING AND PE

1.3.1 Tobacco and Cigarette Smoke

The primary ingredient found within a cigarette is tobacco, dried or cured leaves originating from plants of the nightshade family In addition to the highly addictive alkaloid nicotine, tobacco smoke contains over 4000 chemical

constituents of which nearly 70 have been identified as potent carcinogens '%°

Compounds belonging to the nitrosamine and polycyclic aromatic hydrocarbon groups have been demonstrated to have the largest impact on human cancer risk

245 In depth chemical analysis of tobacco smoke (that which is inhaled) has

identified nicotine, tar and CO as major contributors with exposures of

approximately 0.8, 9 and 12 mg /cigarette, respectively °° The majority of the

compounds present in smoke are formed during combustion, in a pyrolysis- distillation zone (area of chemical decomposition caused by extreme heat) found

just behind the heat-generating combustion zone °' An increased knowledge of

the chemical constituents of cigarette smoke, along with a greater understanding of the negative health impacts caused by these compounds, has led to an introduction of smokeless tobacco products (i.e snuff) into certain populations ?° 3 The majority of smokeless tobacco products contain equivalent levels of nicotine to those measured in cigarettes**’, however exposure to the combustible products of tobacco (i.e tar and CO) is abolished The specific impact(s) of smokeless tobacco on health and disease is currently of significant interest and is a focus of on-going research endeavours

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1.3.2 Smoking and Pregnancy

Maternal cigarette smoking is associated with adverse pregnancy and fetal outcomes (table 1.1); pre-term delivery is increased in women who smoke and their infants are on average 200 g lighter (per pack per day smoked) at birth,

94-96 = Women who smoke in

even after adjustment for gestational age

pregnancy are also two to three times more likely to experience spontaneous abortions compared to non smokers °” °° Smoking also affects placentation with a three to four fold increase in the incidences of placental abruption and placenta previa ‘°° The majority of these adverse outcomes appears to be dose and gestational age dependant and may be reversible or preventable in women who

stop smoking during pregnancy '®' '? While no direct evidence is present concerning which of the 4000 chemical constituents ' found in cigarette smoke

contribute to these poor outcomes, much work has focused on the effects of nicotine, polycyclic aromatic hydrocarbons and thiocyanates, all of which have

been shown to cross the placenta 11° 1.3.3 Smoking and PE

Interestingly, women who smoke cigarettes throughout pregnancy are at a

33% reduced risk of developing PE '°’"'°° Cigarette smoking is associated with

adverse vascular effects in adult tissue, such as hypertension and

atherosclerosis ''° '"’: therefore it is surprising to observe a correlation between

maternal cigarette smoking and a decreased incidence of PE, a vascular disorder It is not fully understood how smoking may reduce the risk of

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developing PE, however there have been several hypothesis put forth Pre- eclampsia has been linked initially to the immune system First time mothers are at a higher risk of developing this disorder and this correlation is directly related

to the length of time the woman had been exposed to her partner’s semen *'

suggesting an acquired immune tolerance that is protective against the development of the disease Women who smoke may have an underlying decreased immune response and may therefore be less likely to develop the

exaggerated immune response seen in PE '* Chronic smoking may also induce

systemic endothelial events which subsequently down regulate endothelial

sensitivity to activation signals (i.e placental debris and ROS) '"* In fact, women

who smoke and who subsequently developed PE have a much poorer outcome than non-smoking women who develop PE which would be consistent with the hypothesis that there is underlying synergy in the endothelial pathology of

smoking and PE ""° In mothers whom the total burden of 'trigger' for systemic

endothelial dysfunction overcomes the desensitization offered by smoking could reasonably be predicted to have more severe utero-placental disease

With every inhalation of cigarette smoke a person is being exposed to thousands of chemicals Therefore, it is challenging to determine which compound(s) may be involved in reducing the risk of developing PE A recent study found that the reduced risk of PE associated with cigarette smoking was not observed in women who used snuff, a form of smokeless tobacco ''* This would indicate that the active compound(s) that is responsible for decreasing the